Original Article
352 Rev Bras Hematol Hemoter. 2012;34(5):352-5
Serum concentrations of nitrite and malondialdehyde as markers of oxidative stress in
chronic myeloid leukemia patients treated with tyrosine kinase inhibitors
Introduction
Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder resulting from neoplastic transformation of primitive hematopoietic cells. It is characterized by a balanced translocation between the long arms of chromosome 9 and 22, which induces formation of the Philadelphia chromosome, resulting from the binding of the BCR gene on chromosome 22 to the ABL gene on chromosome 9, generating the hybrid BCR-ABL gene on chromosome 22q. The product of this oncogene is the bcr-abl protein that has a higher tyrosine kinase activity and is responsible for the pathogenesis of CML(1-3).
Advances in understanding the pathophysiology of the disease are essential for the development of any molecular therapy for CML. Tyrosine kinase inhibitors (TKIs) competitively bind to the receptor for BCR-ABL, ATP-dependent, and inhibit the phosphorylation of tyrosine kinase, thereby avoiding a change in conformation to the active form and normalizing the mechanisms that regulate cell proliferation with the inhibition of cell proliferation and induction of leukemic apoptose(3-6). Imatinib, a TKI, has revolutionized the treatment of CML. Despite the enduring responses of imatinib, some chronic phase patients and a higher proportion of advanced phase patients exhibit intolerance or resistance to this medication(7). Primary resistance occurs when there is failure to achieve a significant hematologic or cytogenetic response by the patient from the beginning of treatment. Secondary resistance is the resurgence of the leukemic clone after an initial response to the drug(7,8). To treat patients who are resistant or intolerant to imatinib, a new generation of BCR-ABL TKIs has been developed, including dasatinib and nilotinib(9,10).
The mechanism whereby the (9:22) translocation occurs is unknown, although some studies suggest that oxidative stress may be involved in the genesis of CML(11-13). Moreover, BCR-ABL positive cells are a source of reactive oxygen species (ROS), which cause oxidative DNA damage and may contribute to the formation of additional abnormalities leading to disease progression to the more advanced stages (accelerated and blast crisis phases) or resistance to TKIs such as imatinib(14-16).
The present study aimed to investigate markers of oxidative stress in CML patients treated with TKIs.
Maria Juracy Petrola1
Alana Joselina Montenegro de Castro1 Maria Helena da Silva Pitombeira1 Maritza Cavalcante Barbosa1 Acy Telles de Souza Quixadá2 Fernando Barroso Duarte1 Romelia Pinheiro Gonçalves1
1Universidade Federal do Ceará - UFC, Fortaleza, CE, Brazil
2Hospital Universitário Walter Cantídio - HUWC, Fortaleza, CE, Brazil
Conlict-of-interest disclosure:
The authors declare no competing inancial interest
Submitted: 3/27/2012 Accepted: 6/15/2012
Corresponding author: Romélia Pinheiro Gonçalves
Faculdade de Farmácia da Universidade Federal do Ceará - UFC
Rua Pereira Valente, 640, apto 701, Meireles 60160-250 Fortaleza, CE, Brazil
Phone: 55-85-87879459 [email protected]
www.rbhh.org or www.scielo.br/rbhh DOI: 10.5581/1516-8484.20120091
Background: Chronic myeloid leukemia is a neoplasm characterized by clonal expansion of hematopoietic progenitor cells resulting from the (9:22)(q34,11) translocation. The tyrosine kinase abl fusion protein, the initial leukemogenic event in chronic myeloid leukemia, is constitutively activated thus inducing the production of reactive oxygen species. Of particular relevance is the fact that an increase in reactive oxygen species can facilitate genomic instability and may contribute to disease progression.
Objetive: To evaluate oxidative stress by determining the levels of malondialdehyde and nitrite in chronic myeloid leukemia patients under treatment with 1st and 2nd generation tyrosine kinase inhibitors monitored at a referral hospital in Fortaleza, Ceará.
Methods: A cross-sectional study was performed of 64 male and female adults. Patients were stratiied according to
treatment. The levels of malondialdehyde and nitrite were determined by spectrophotometry. Statistical differences between groups were observed using the Student t-test and Fisher’s exact test. The results are expressed as mean ±
standard error of mean. The signiicance level was set for a p-value < 0.05 in all analyses.
Results: The results show signiicantly higher mean concentrations of nitrite and malondialdehyde in
chronic myeloid leukemia patients using second-generation tyrosine kinase inhibitors compared to patients on imatinib.
Conclusion: It follows that chronic myeloid leukemia patients present higher oxidative activity and that the increases in oxidative damage markers can indicate resistance to 1st generation tyrosine kinase inhibitors.
353 Serum concentrations of nitrite and malondialdehyde as markers of oxidative stress in chronic myeloid leukemia patients treated with tyrosine kinase inhibitors
Rev Bras Hematol Hemoter. 2012;34(5):352-5 Methods
This is a cross-sectional study of 64 adult patients diagnosed with CML according to the criteria of the World Health
Organization (WHO) classiication(17), treated in the hematology service of a referral hospital in Fortaleza, Ceará.
The group of patients consisted of 34 women (53.1%) and 30 men (46.9%), with ages that ranged from 20 to 80 years and
with a median age of 44 years. The patients were stratiied into
two groups according to treatment: imatinib (n = 31) and second-generation TKIs (dasatinib and nilotinib - n = 33). Patients were excluded if they were smokers, drank alcohol, were drug addicts or took vitamins with antioxidant action.
The level of malondialdehyde (MDA) in heparinized plasma was calculated by determining the quantity of reactive substances using thiobarbituric acid (TBARS) at a temperature of 100°C. The absorbance of the colored MDA-TBARS complex was measured by spectrophotometry at a wavelength of 560 nm(18). Nitrite levels in heparinized plasma were determined using Green’s method where nitrite in an acid medium reacts with sulfanilamide. The resulting diazo compound reacts with N-naphthyl-ethylenediamine (NEED), thereby generating a compound with an intense red color. Absorbance was measured by spectrophotometry at a wavelength of 540 nm(19).
All subjects signed written consent forms and the study was approved by the Research Ethics Committee of the Universidade Federal do Ceará, under protocol number 042.05.10 which is in accordance with Resolution 196/96 of the National Health Council.
The GraphPrism (version 5.01) program was used for statistical analysis. The Kolmogorov-Smirnov test was used to check for normal distribution of the data. Statistical differences
between groups were identiied using the Student t-test and Fisher’s exact test with the signiicance level being set for a
p-value < 0.05 in all analyses. The results are expressed as means ± standard error of mean (SEM).
Results
The clinical characteristics of patients in the study are shown
in Table 1. There were no signiicant differences for gender,
current age, age at diagnosis and time since diagnosis between the groups (p-value > 0.05).
Of the 33 patients treated with second-generation TKIs, 15
(44.5%) had high Sokal risk at diagnosis, whereas this inding was observed in ive patients (16.1%) in the group treated with
imatinib (p-value < 0.05). In relation to disease progression, 21 patients (63.6%) treated with second-generation TKIs were in the accelerated or blast crisis phases, while this was observed in six patients (19.5%) treated with imatinib (p-value < 0.05).
The mean level of nitrite in the plasma of CML patients was 2.638 ± 0.327 mM and the mean level of MDA was 3.582 ± 0.306 mM. On stratifying the CML patients according to treatment, it was observed that serum levels of nitrite and MDA were
signiicantly higher in patients treated with second-generation
TKIs than those treated with imatinib (Figure 1 and Table 1).
Discussion
CML accounts for approximately 14% of all leukemias with an annual incidence of one to two cases per 100,000 inhabitants and a slight predominance of men(20,21). It affects all ages, but the incidence rises with age(21). The average age at diagnosis is 55 to 60 years, with less than 10% of patients being younger than 20 years old(21,22). In the present study, no signiicant difference in the incidence of the disease between genders was found, however there was a slight predominance of males, which agrees with published data. The mean age at diagnosis was 44 years in the
Table 1 - Analysis of measurements of Imatinib and 2nd-generation tyrosine kinase inhibitors Groups on day D-1
Parameter CML (n = 64) Imatinib (n = 31) 2nd-generation (n = 33) p-value
Gender (male/female) 34/30 16/15 18/15 0.5062
Age (years) - mean ± SE 45.30 ± 2.08 43.92 ± 2.99 46.63 ± 2.93 0.2609a
Age at diagnosis (years) - mean ± SE 44.22 ± 2.01 41.64 ± 2.78 46.17 ± 2.80 0.1345a
Time since diagnosis (years) - mean ± SE 7.755 ± 0.54 7.200 ± 0.99 8.778 ± 0.86 0.1296a
High Sokal risk – n (%) 20 (31.2) 5 (16.1) 15 (44.5) 0.0215b
Accelerated phase + blast crisis - n (%) 27 (42.2) 6 (19.5) 21 (63.6) 0.0014b
Nitrite (µM) - mean ± SE 2.638 ± 0.327 2.010 ± 0.449 3.730 ± 0.429 < 0.0001a
MDA (µM) - mean ± SE 3.582 ± 0.306 2.818 ± 0.349 4.197 ± 0.445 < 0.01a
CML: chronic myeloid leukemia; MDA: malondialdehyde a Student t-test; b Fisher’s exact Test; statistically signiicance < 0.05
Figure 1 - Nitrite and malondialdehyde levels in chronic myeloid leukemia patients according to treatment
354
Petrola MJ, Castro AM, Pitombeira MH, Barbosa MC, Quixadá AT, Duarte FB, Gonçalves RP
Rev Bras Hematol Hemoter. 2012;34(5):352-5 study patients. In recent years, there has been a decrease in mean
age at diagnosis of CML. Studies in India, Pakistan and Brazil showed mean ages of 38, 35 and 41 years, respectively. This fact may be attributed to the occurrence of widespread routine screening, as well as genetic factors, environmental factors and public health(23,24).
The prognosis of CML may vary signiicantly even in
patients who are at the same stage of the disease. Strategies for
risk stratiication are important as a guide for the prognosis and
treatment of patients. The Sokal score is the most widely used
and has prognostic signiicance for factors such as age over
60 years, spleen size, platelet count above 700 x109/L and the number of basophils and blasts in peripheral blood and bone marrow(25,26). In the present study, most patients treated with second-generation TKIs had high Sokal risk at diagnosis and were in advanced stages of the disease at the time of the study.
This inding suggests that the Sokal score, although it was
created for patients on drugs that are no longer used to treat this disease, retains its predictive value after the advent of TKIs to treat CML as has been reported in recent studies(27).
Markers of cell damage are being studied in order to elucidate the role of oxidative stress in CML(28). These include the free radical, nitrite (NO2-), which is associated to direct damage of cellular components and MDA, which relects the
extent of lipid peroxidation and modulates the expression of genes related to promoting tumor progression. Previous studies have shown severe oxidative stress in CML and other hematological cancers(29-35).
Imatinib has been used as irst-line therapy for CML
providing lasting responses in most patients, especially those in the chronic phase. Even so some in the chronic phase and a higher proportion of patients in the more advanced stages of CML are resistant or intolerant to imatinib(36). Mechanisms of resistance to imatinib can be classiied as BCR-ABL independent or dependent. The irst group includes the binding of imatinib-α 1-acid glycoprotein, increased expression of drug eflux pumps and reduced expression of drug inlux
transporters. The BCR-ABL dependent mechanisms are increased expression of the oncoprotein and point mutations in the tyrosine kinase domain. These are the most common occurrences in imatinib resistance(8,37,38).
Second-generation TKIs (dasatinib and nilotinib) are inhibitors of multiple targets, able to inhibit the active and inactive forms of the bcr-abl tyrosine kinase protein as well as Src family kinases. In addition, second-generation TKIs have been shown to be active against all the mutations of the Bcr-Abl oncoprotein resistant to imatinib except T315I(8,37-39).
In the present study, patients treated with second-generation
TKIs had signiicantly higher levels of nitrite and MDA when
compared to patients treated with imatinib. Such results suggest that oxidative stress parameters can be used to predict lack of response to 1st generation TKIs. Patients who are refractory to imatinib had higher levels of oxidative damage markers, possibly due to a high rate of additional mutations, the leading causes of
resistance to irst-line therapy. Studies show that the occurrence of
mutations is closely associated with oxidative stress and disease progression(39). Therefore, it is likely that reactive oxygen species
(ROS) participate in combination with the BCR-ABL gene by introducing new mutations in the fusion protein, including those that cause resistance to imatinib(40,41).
The results of this study suggest that high levels of oxidative stress may be associated with mutations that cause resistance
to irst-line treatment. Moreover, high levels of oxidative stress
markers are associated with the group of patients with high Sokal scores suggesting that this score may also be useful as a prognostic tool to identify patient’s response to imatinib. However, detailed studies are needed to identify the origin of oxidative stress and the ROS produced by cells in BCR-ABL to better understand the role and prognostic impact of elevated levels of ROS in CML.
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